BASIC METABOLIC PATHWAYS

Introduction: Metabolism

The entire spectrum of chemical reactions, occurring in the living system is

collectively referred to as metabolism.
A metabolic pathway constitutes a series of enzymatic reactions to produce
specific products.

Metabolism is broadly divided into two categories:

Metabolism

Catabolism Anabolism

-->Degradative/Breakdown -->Synthesis/formation
processes processes
-->Complex molecules to -->simpler ones to Complex
simpler ones molecules
-->Release of energy -->Utilization of energy

Photosynthesis
Green plants carry out ‘photosynthesis’, a physicochemical process by which
they use light energy to drive the synthesis of organic compounds.
Photosynthesis is important due to two reasons: it is the primary source of all
food on earth. It is also responsible for the release of oxygen into the
atmosphere by green plants.

Plants could use light energy to make carbohydrates from CO 2 and water.
The empirical equation representing the total process of photosynthesis is:
CO2 + H2O, light (CH2O) + O2
 The overall process of photosynthesis is therefore:
6CO2 + 12H2O, light  C6H12O6 + 6H2O + 6O2

The chloroplast
The photosynthesis takes place in chloroplast.
Chloroplast is consists of membranous system.
Membranous system consisting of grana, the stroma lamellae, and the matrix
stroma.
The membrane system is responsible for trapping the light energy and also for
the synthesis of ATP and NADPH.
In stroma, enzymatic reactions synthesise sugar, which in turn forms starch.
The former sets of reactions, since they are directly light driven are called
light reactions (photochemical reactions). The latter are not directly light
driven but are dependent on the products of light reactions (ATP and
NADPH). Hence, to distinguish the latter they are called as dark reactions
(carbon reactions).

Light Reaction
Light reactions/Photochemical phase include
 light absorption,
 water splitting,
 oxygen release, and
  the formation of high-energy chemical intermediates (ATP & NADPH)
Several protein complexes are involved in the process.
The pigments are organised into two discrete photochemical light harvesting
complexes (LHC) within the Photosystem I (PS I) and Photosystem II (PS
II).
The LHC are made up of hundreds of pigment molecules bound to proteins.
Each photosystem has all the pigments (except one molecule of chlorophyll-a)
forming a light harvesting system also called antennae.
These pigments help to make photosynthesis more efficient by absorbing
different wavelengths of light. The single chlorophyll-a molecule forms the
reaction centre.
The reaction centre is different in both the photosystems. In PS I the reaction
centre chlorophyll-a has an absorption peak at 700 nm, hence is called P700,
while in PS II it has absorption maxima at 680 nm, and is called P680.

The Electron Transport

In PS-II the reaction centre chlorophyll-a absorbs 680 nm wavelength of red
light causing electrons to become excited and jump into an orbit farther from
the atomic nucleus. These electrons are picked up by an electron acceptor
which passes them to an electrons transport system consisting of
cytochromes. This movement of electrons is downhill, in terms of an
oxidation-reduction or redox potential scale.
The electrons are not used up as they pass through the electron transport
chain, but are passed on to the pigments of photosystem PS-I.
Simultaneously, electrons in the reaction centre of PS-I are also excited when
they receive red light of wavelength 700 nm and are transferred to another
accepter molecule that has a greater redox potential.
These electrons then are moved downhill again, this time to a molecule of
energy-rich NADP+. The addition of these electrons reduces NADP+ to
NADPH + H+.
This whole scheme of transfer of electrons, starting from the PS-II, uphill to
the acceptor, down the electron transport chain to PS-I, excitation of
electrons, transfer to another acceptor, and finally downhill to NADP+ reducing
it to NADPH + H+ is called the Z scheme, due to its characteristic shape. This
 shape is formed when all the carriers are placed in a sequence on a redox
potential scale.

Water Molecule Splitting

The electrons that were moved from PS-II must be replaced. This is achieved
by electrons available due to splitting of water.
The splitting of water is associated with the PS-II; water is split into 2H+, [O]
and electrons. This creates oxygen, one of the net products of
photosynthesis.
The electrons needed to replace those removed from PS-I are provided by
PPS-II.
2H2O  4H+ + O2 + 4e-

 The products of light reaction are ATP, NADPH and O2, of these O2 diffuses
out of the chloroplast while ATP and NADPH are used to drive the processes
leading to the synthesis of food, more precisely, sugars. This is the
biosynthetic phase of photosynthesis. This process does not directly depend
on the presence of light but is dependent on the products of the light reaction,
i.e., ATP and NADPH, besides CO2 and H2O.
  Immediately after light becomes unavailable, the biosynthetic process
continues for some time, and then stops. If then, light is made available, the
synthesis starts again.
14
Melvin Calvin by using C discovered the first CO2 fixation product as 3-
carbon organic acid. He also contributed the complete biosynthetic pathway;
hence it was called Calvin cycle after him. The first product identified was 3-
phosphoglyceric acid (3-PGA).
Based on experiments another group of plants were discovered, where the first
stable product of CO2 fixation was an organic acid, which had 4 carbon atoms in it.
This acid was identified to be oxalo-acetic acid (OAA). Since then CO2 assimilation
during photosynthesis was said to be of two main types: those plants in which the
first product of CO2 fixation is a C3 acid (PGA), i.e., the C3 pathway, and those in
which the first product was a C4 acid (OAA), i.e., the C4 pathway.

The Calvin Cycle

The Calvin cycle can be described under three stages:

a) Carboxylation,
b) Reduction and
c) Regeneration.
1. Carboxylation: Carboxylation is the fixation of CO2 into a stable organic
intermediate. In Carboxylation step, CO2 is utilised for the carboxylation of ribulose-
1,5-bisphosphate (RuBP). This reaction is catalysed by the enzyme RuBP
carboxylase which results in the formation of two molecules of 3-PGA.

2. Reduction: These are a series of reactions that lead to the formation of glucose.
The steps involve utilisation of 2 molecules of ATP for phosphorylation and two of
NADPH for reduction per CO2 molecule fixed. The fixation of six molecules of CO2
and 6 turns of the cycle are required for the formation of one molecule of glucose
from the pathway.

3. Regeneration: Regeneration of the CO2 acceptor molecule RuBP is crucial if the

cycle is to continue uninterrupted. The regeneration steps require one ATP for
phosphorylation to form RuBP.
Hence for every CO2 molecule entering the Calvin cycle, 3 molecules of ATP
and 2 of NADPH are required. It is probably to meet this difference in number
of ATP and NADPH used in the dark reaction that the cyclic phosphorylation
takes place.
To make one molecule of glucose 6 turns of the cycle are required.
In Out
Six CO2 One glucose
18 ATP 18 ADP
12 NADPH 12 NADP
 INTRODUCTION TO BIOGENESIS

The living plant may be considered as a biosynthetic laboratory not only for
the primary metabolites like sugars, amino acids and fatty acids that are
utilized as food by man, but also for a multitude of secondary products of
pharmaceutical significance such as glycosides, alkaloids, flavonoids, volatile
oils, resins, tannins, terpenoids, etc.
A higher plant is a solar-powered biochemical factory which manufactures
both primary and secondary metabolites from air, water, minerals and
sunlight.
Primary metabolites are substances that are widely distributed in nature,
occurring in one form or another in virtually all organisms and are needed for
general growth and physiological development, because of their basic cell
metabolism.
Secondary metabolites are biosynthetically derived from primary metabolites
but are more limited in distribution, usually being restricted to a taxonomic
group. They may represent chemical adaptations to environmental stresses,
or they may serve as defensive, protective or offensive chemicals against
microorganisms, insects and higher herbivorous predators.
They are sometimes considered to be waste or secretory products of plant
metabolism. In terms of cellular economy, secondary products are in general
expensive to produce and accumulate, and are, therefore frequently present
in plants in much smaller quantities than the primary metabolites.
The major source of carbon is usually glucose, which is photosynthesized in
green plants.
The recent advances in the field of biochemistry have greatly clarified
enzyme-catalysed and interrelated reactions resulting in formation of primary
metabolites and their role in the synthesis of secondary products, many of
which are used by man.
Carbon metabolism in plants
The basic carbon reduction cycle by which carbon dioxide is converted to
sugar phosphates is of primary importance, both as an energy yielding
process and also as a source of various metabolic intermediates.
Two biosynthetic pathways of special importance in breakdown of sugar are
pentose phosphate cycle (direct pathway) and glycolysis. The hexose
phosphate is oxidized first into carbon dioxide and pentose phosphate in
direct pathway.
The pentose phosphate formed due to biosynthetic degradation may then be
utilized as such or otherwise converted by a series of metabolic reactions into
triose phosphate or by a reversal of glycolysis, into a hexose; however, in
glycolysis, hexose phosphate is split hydrolytically to yield triose phosphate,
which can then be oxidized. Accumulation of knowledge in biogenetic field
makes it possible to predict the gross biogenetic origin of practically all plant
products.

Flowchart of some cell metabolites

Glycolysis
HMP shunt
 Shikimate - Argenate Pathway
A Shikimic acid pathway is an important route from carbohydrate for the biosynthesis
of the C6–C3 units (phenylpropane derivatives), of which phenylalanine and tyrosine
are both examples. A scheme of biogenesis for these aromatic amino acids, as
elucidated in various organisms, for higher plants, the presence of the enzyme
system responsible for the synthesis of shikimic acid has been confirmed.

An important branching point arises at chorismic acid; anthranilate synthase uses

chorismic acid as a substrate to give anthranilic acid which is a precursor of
tryptophan. The synthesis is controlled by the latter acting as a feedback inhibitor;
chorismate mutase converts chorismic acid to prephenate, the precursor of
phenylalanine and tyrosine, and a variety of control mechanisms appear to operate
at the branching point.

The opium alkaloids are synthesized via this pathway and two isoforms of
chorismate mutase have been isolated and characterized from poppy seedlings.
Although there are only small differences in the sequence of reactions for the
shikimate pathway in bacteria, fungi and plants there are considerable differences in
the molecular organization of the pathway.

The Shikimic acid is a key intermediate from carbohydrate for the biosynthesis of C6
– C3 units (phenylpropane derivatives).

The shikimic acid pathway is also important in the genesis of the aromatic building
blocks of lignin and in the formation of some tannins, vanillin and phenylpropane
units of the flavones and coumarins.
E-1: DAHP Synthase
E-2: Dehydroquinate Synthase
E-3: Dehydroquinate- Shikimate Dehydratase
E-4: Dehydroquinate- Shikimate Dehydrogenase
E-5: Shikimate kinase
E-6: EPSP Synthase
E-7: Chorismate Synthase
E-8: Chorismate mutase
E-9: Prephenate aminotransferase
E-10: Arogenate dehydrogenase
E-11: Arogenate dehydratase
E-12: Anthranilate Synthase
E-13: Phosphoribosyl pyrophosphate (PRPP)-Anthranilate transferase
E-14: Phosphoribosyl pyrophosphate-Anthranilate isomerase
E-15: Indole-3-glycerol phosphate Synthase
E-16: Tryptophan synthase-α
E-17: Tryptophan synthase-β
 Origins of some secondary metabolites in relation to the basic metabolic
pathways

IPP-isopentenyl diphosphate; DOX-deoxyxylulose; MVA- mevalonic acid

The acetate hypothesis
Acetate is the produced from pyruvate by decarboxylation and dehydrogenation
reaction, in which pyruvate dehydrogenase complex, multiple enzyme system plays
a major role.

Acetate-Mevalonate pathway
 Amino acid pathway
Amino acids occur in plants both in the Free State and as the basic units of proteins
and other metabolites. They are compounds containing one or more amino groups
and one or more carboxylic acid groups.

Most of those found in nature are α-amino acids with an asymmetric carbon atom
and the general formula R–CH-(NH2)-COOH. Some 20 different ones have been
isolated from proteins, all having an l-configuration. Other amino acids occur in the
Free State and some having the d-configuration have been isolated from plants and
microorganisms, where they may form antibiotic polypeptides.

Many amino acids contain only carbon, hydrogen, oxygen and nitrogen, but other
atoms may be present (e.g. sulphur in cystine, and iodine in thyroxin). In case of
some amino acids, more than one amino group (e.g. lysine, diaminocaproic acid)
and more than one carboxylic acid group (e.g. aspartic or aminosuccinic acid) may
be present. Some amino acids are aromatic such as phenylalanine, or heterocyclic
such as proline (pyrrolidine nucleus), tryptophan (indole nucleus) and histidine
(imidazole nucleus).

Biosynthesis of amino acids

As amino acids are also the precursors of some secondary metabolites, their
biosyntheses will be arising at various levels of the glycolytic and TCA systems.

Nitrogen appears to enter the metabolism of the organism by reductive amination of

α-keto acids; pyruvic, oxaloacetic and α-ketoglutaric acids give alanine, aspartic acid
and glutamic acid, respectively.
By transamination reactions with other appropriate acids, alanine, aspartic acid and
glutamic acid serve as α-amino donors in the formation of other amino acids. The
general transamination reaction may be written:

R-CH-(NH2)-COOH + R’-CO-COOH ⇔ R-CO-COOH + R’-CH(NH2)-COOH

Glutamic acid, in particular, appears to be a central product in amino acid
metabolism and glutamic acid dehydrogenase has been reported in a number of
plant tissues. The enzyme functions in conjunction with NAD:

α-ketoglutaric acid + NH3 +NADPH ⇔ glutamic acid+ NAD

That the nitrogen of ammonia first appears in the dicarboxylic amino acids and is
later transferred to other nitrogen compounds was demonstrated in some of the
15
earliest plant biochemistry experiments, which utilized N.

Proline, hydroxyproline, ornithine and arginine

These amino acids are of importance in the secondary metabolism of some plants in
that they are precursors of a number of alkaloids. They are metabolically connected
to glutamic acid and their formation in plant cells is complex in that the reactions are
strictly compartmentalized. The enzymes involved have been characterized, and for
the formation of ornithine it is the N-acetyl derivatives which are involved. Arginine
appears to be synthesized from ornithine in all organisms via the reactions of the
urea cycle.

Serine and glycine

Together with cysteine and cystine, these amino acids arise at the triose level of
metabolism.
Serine and glycine are readily interconvertible:

In animal tissues it has been shown that tetrahydrofolic acid (THFA) is responsible
for the removal of the hydroxymethyl group of serine to form hydroxymethyltetrafolic
acid. As this compound serves as a source of formate and methyl groups in many
reactions, the β-carbon of serine may be their original source; this applies to the
formation of methionine, itself an important methyl donor in plant biochemistry.

Alanine, valine and leucine

Studies with microorganisms and yeasts have shown these amino acids to be
derived from pyruvate. There is evidence that α-ketoisovaleric acid is aminated to
form valine and that it can also condense with acetate to form an intermediate which
on decarboxylation and amination affords leucine.
Isoleucine
This amino acid is formed by a similar series of reactions to valine but commencing
with α-aceto-α-hydroxypropionic acid instead of α-acetolactic acid.
Lysine
Lysine, H2N–(CH2)4–CH(NH2)–COOH, is derived, in plants, from aspartate involving
a pathway utilizing 2,3-dihydropicolinic acid and diaminopimelic acid. It is the
precursor of some alkaloids of Nicotiana, Lupinus and Punica.

Aromatic amino acids

These have already been mentioned in the discussion of the biosynthesis of
aromatic compounds.
 Study of utilization of radioactive isotopes in the investigation of
Biogenetic studies
The elucidation of biosynthetic pathways in plants for the production of various plant
metabolites has been extensively examined by means of isotopically labelled
precursors. With the advancement of tracer technology, it is possible to incorporate
isotopes into presumed precursors of plant metabolites and use as markers in
biogenetic experiments. With the use of radioactive carbon ( 14C) and hydrogen (3H)
and to a lesser extent sulphur (36S) and phosphorous (32P), it is possible to
understand various biosynthetic pathways. A labelled nitrogen atom may give more
specific information about the biosynthesis of alkaloids, proteins and amino acids.

The most notable success by use of isotopically labelled precursors was achieved by
Birch in the biosynthetic investigations of mould metabolites such as 6-
14
methylsalicylic acid and griseofulvin from C-labelled acetate.

Biosynthesis: Formation of a chemical compound by a living organism.

Biogenesis: Production or generation of living organisms from other living

organisms.

Various intermediate and steps are involved in biosynthetic pathway in plants can be
investigated by means of following techniques:

 Tracer techniques
 Use of isolated organs or tissues
 Grafting method
 Use of mutant strains
 Enzymatic studies

Tracer techniques:
A technique which utilizes a labelled compound to trace or find out the different
intermediates and various steps involved in biosynthetic process in plant at given
rate and given time. When these labelled compounds are administered into the
plants, they become a part of general metabolic pool and undergo reactions
characteristic to the metabolism of that particular plant.

Basic steps involved in tracer techniques:

1. Preparation of labeled compound
2. Incorporation of labeled compound to tissue system
3. Separation or isolation of labeled compound from tissue system
4. Determination of nature of metabolites in various biochemical fraction

1. Preparation of labeled compound:

14
i. The labelled compound produces by growing chlorella in atmosphere of CO2.
Hence, all carbon compounds 14C labelled.
ii. The 3H (tritium) labelled compound are commercially available. Tritium labelling
is effected by catalytic exchange (Pt) in aqueous media by hydrogenation of
unsaturated compound with tritium gas. Tritium is pure β-emitter of low intensity
14
and its radiation energy is lower than C.
iii. By the use of organic synthesis:
CH3MgBr + 14CO2 CH314COOHMgBr + H2O

CH314COOHMgBr  CH314COOH + Mg-(OH)Br

The labelled compounds may be prepared by use of two types of isotopes:

 Radioactive isotopes: Decay with emission of radiation. E.g: 3H, 14

C, 24
Na,
42
K, 35S, 32P, 131I, 60Co.
– For biological investigation – carbon & hydrogen.

– For metabolic studies – S, P, and alkali and alkaline earth metals are
used.

– For studies on protein, alkaloids, and amino acid – labelled nitrogen

atom give more specific information.

– 3H compound is commercially available.

• Stable isotopes: e.g. 2H, 13C, 15N, 18O

– Used for labelling compounds as possible intermediates in
biosynthetic pathways.
 – Usual method of detection are: – Mass spectroscopy [15N, 18O], NMR
spectroscopy [2H, 13C]

Properties of some radioactive isotopes:

Natural Radioactive Radiation Half life
C12 C14 beta 5760 yrs
H1 H3 beta 12.5 yrs
S32 S35 beta 871 days
P31 P32 beta 14.3 days
Cl35 Cl36 beta 4.4 × 105yrs
I127 I131 Beta, Gamma 8 days
Co59 Co60 Beta, Gamma 5.3 days

2. Introduction of labeled compound to tissue system:

There are six methods use to incorporate labelled compound to tissue system:
a. Root feeding: The plant in which roots are the biosynthetic sites, this method
is preferred. E.g. Tobacco. In this type of experiment the plant are cultivated
hydroponically to avoid microbial contamination.
b. Stem feeding: Presence of root don’t require for biosynthesis. In this method
substrate can be administered through the cut ends of stem immersed in a
solution. For Latex containing plant this method is not suitable.
c. Direct injection: This method is applicable to the plant with hollow stem. E.g.
Umbelliferae and capsule bearing plant (opium poppy).
d. In-filtration/Wick feeding: When it is desired to carryout feeding on plant
rooted in soil or other support without disturbing the root, wick feeding is
applicable.
e. Floating method: When small amount of material is available, floating method
is used. In this method, leaf disc or chopped leaves are floating on the
substrate solution. This technique is also used in conjugation with vacuum
infiltration to remove gases.
f. Spray technique: In this method compound have been absorbed after being
sprayed on leaves in aqueous solution. E.g. Steroids.
3. Separation or isolation of labeled compound or metabolite:
Depending on the nature of drug and its source different method of extraction is
employed.

 Soft and fresh tissue: Infusion, maceration

 Hard tissue: Decoction and Hot percolation
 Unorganized drug: Maceration with adjustment
Choice of solvent for extraction:

 Fat and oil: non polar solvent

 Alkaloid, Glycoside, Flavonoid: slightly polar solvent
 Plant phenol: Polar solvent
Fractional crystallization, partition, column chromatography also used as separation
technique.

4. Determination of nature of metabolites:

Depending on nature of isotopes various instrumentation techniques is used for
determination of chemical nature of intermediate and final product.

For radioactive isotopes,

 Geiger – Muller counter
 Scintillation or liquid scintillation counter
 Ionization chamber
 Bernstein – Bellentine counter.
These entire instruments characterize the nature of radiation. Basically it depends
upon the conversion of kinetic energy of particle into fleeting pulse of light as a result
of its penetration into a suitable luminescent medium.

For stable isotopes,

 Mass spectroscopy- gives molecular peaks depending on mass/charge ratio.
 NMR- gives nature of carbon and proton.
 Autoradiography.
 Radio paper chromatography
Methods in Tracer Techniques
i. Precursor- Product Sequence:
For elucidation of biosynthetic pathways in plants by means of labeled compounds,
the precursor product sequence method is used. In this method presumed precursor
of the constituent is under investigation in a labeled form is fed to plant and after a
particular time the constituent is isolated, purified and its radioactivity is determined.

Application:
• Stopping of hordenine production in barley seedling after 15-20 days of
germination.
• Restricted synthesis of hyoscine, distinct from hyoscyamine in Datura
stramonium.
• This method is applied to the biogenesis of morphine & ergot alkaloids.
Disadvantage: The radioactivity of isolated compound alone is not usually sufficient
evidence that the particular compound fed is direct precursor, because substance
may enter the general metabolic pathway and from there may become randomly
distributed through a whole range of product.

ii. Double & multiple labelling:

This method give the evidence for nature of biochemical incorporation of precursor
arises double & triple labelling. In this method specifically labelled precursor and
their subsequent degradation of recover product are more employed.

Application: -
 This method is extensively applied to study the biogenesis of plant secondary
metabolite.
 Used for study of morphine alkaloid.
E.g. Leete, use doubly labeled lysine used to determine which hydrogen of lysine
molecule was involved in formation of piperidine ring of anabasine in Nicotina
glauca.
 iii. Competitive feeding: This method is normally used to determine two
possible intermediates in the plants.
Competitive feeding can distinguish whether B or B1 is the normal intermediate in the
formation of C from A.

OR

Application:
• This method is used for elucidation of biogenesis of propane alkaloids.
• Biosynthesis of hemlock alkaloids (coniine, conhydrine etc) e.g. biosynthesis
of alkaloids of Conium maculactum (hemlock) using 14C labelled compounds.
iv. Isotope Incorporation: This method provides information about the position of
bond cleavage & their formation during reaction.

E.g. Glucose-1-Phosphatase cleavages as catalysed by alkaline phosphatase, this

reaction occur with cleavage of either C – O bond or P – O bond.

v. Sequential Analysis:
The principle of this method of investigation is to grow plants in the atmosphere of
14
CO2 and by analysis of plants at a given time intervals, to obtained the sequence
in which various related compounds becomes labeled from the results some
biosynthetic route may be accepted and others rejected.

Application:
 14
CO2 and sequential analysis has been very successfully used in elucidation
of carbon in photosynthesis.
  Determination of sequential formation of opium hemlock and tobacco
alkaloids.
 Exposure as less as 5 min. 14
CO2, is used in detecting biosynthetic sequence
as
Piperitone(-)-Menthone (-)-Menthol in Mentha piperita.

Application of Tracer Technique

14
• Study of squalene cyclization by use of C, 3H labelled mevalonic acid.
• Interrelationship among 4-methyl sterols and 4, 4-dimethyl sterols, by use of
14
C acetate.
• Terpenoid biosynthesis by chloroplast isolated in organic solvent, by use of 2-
14
C mevalonate.
• Study the formation of cinnamic acid in pathway of coumarin from labelled
coumarin.
14 15
• Origin of carbon & nitrogen atoms of purine ring system by use of C or N
labelled precursor.
• Study of formation of scopoletin by use of labelled phenylalanine.
45
• By use of Ca as tracer, found that the uptake of calcium by plants from the
soil. (CaO & CaCO3).
32
• By adding ammonium phosphate labelled with P of known specific activity
the uptake of phosphorus is followed by measuring the radioactivity as label
reaches first in lower part of plant, than the upper part i.e. branches, leaves
etc.